Prevalent mechanism of membrane bridging by synaptotagmin-1

Synaptotagmin-1 functions as a Ca²⁺ sensor in neurotransmitter release through its two C² domains (the C₂A and C ₂B domain). The ability of synaptotagmin-1 to bridge two membranes is likely crucial for its function, enabling cooperation with the soluble Nethylmaleimide sensitive factor adaptor protein receptors (SNAREs) in membrane fusion, but two bridging mechanisms have been proposed. A highly soluble synaptotagmin-1 fragment containing both domains (C₂AB) was shown to bind simultaneously to two membranes via the Ca²⁺-binding loops at the top of both domains and basic residues at the bottom of the C₂B domain (direct bridging mechanism). In contrast, a longer fragment including a linker sequence (lnC₂AB) was found to aggregate in solution and was proposed to bridge membranes through trans interactions between lnC ₂AB oligomers bound to each membrane via the Ca²⁺-binding loops, with no contact of the bottom of the C₂B domain with the membranes. We now show that lnC₂AB containing impurities indeed aggregates in solution, but properly purified lnC₂AB is highly soluble. Moreover, cryo-EM images reveal that a majority of lnC₂AB molecules bridge membranes directly. Fluorescence spectroscopy indicates that the bottom of the C₂B domain contacts the membrane in a sizeable population of molecules of both membranebound C₂AB and membrane-bound lnC₂AB. NMR data on nanodiscs show that a fraction of C ₂AB molecules bind to membranes with antiparallel orientations of the C₂ domains. Together with previous studies, these results show that direct bridging constitutes the prevalent mechanism of membrane bridging by both C₂AB and lnC₂AB, suggesting that this mechanism underlies the function of synaptotagmin-1 in neurotransmitter release.